Thyroxin and triiodothyronine are hormones secreted by the thyroid gland. They enter the bloodstream where they are reversibly bound by plasma proteins. Numerous diseases, most of which involve the thyroid gland, cause abnormal levels of these hormones in the blood plasma. Determination of the quantity of these hormones in the blood plasma provides information useful in the diagnosis and treatment of diseases which affect the levels of these materials in the blood plasma.
The thyroid hormones in blood serum have been measured by competitive protein binding. For example, thyroxin extracted from a measured quantity of blood serum and a measured quantity of thyroxin labeled with either a radioactive element or with a fluorescent material are permitted to react with and come to equilibrium with a measured quantity of antibody to thyroxin. Thyroxin bound to the antibody is separated from thyroxin not bound to the antibody and the amount of labeled thyroxin in either the bound or unbound thyroxin portions is determined from the amount of labeled thyroxin found in either the bound or unbound portions of the product. The quantity of thyroxin in the serum can then be determined.
Heretofore, most thyroid hormone analyses have been made using thyroxin labeled with radioactive iodine by the competitive protein binding method. Fluorescent assays are superior to radio assay in several respects, e.g., personal safety, low capital investment and stability of materials. The adoption of fluorescent assay methods has been impeded by unavailability of suitable fluorescent labeled thyroxin, adequate separation of bound from unbound thyroxin and insufficiently sensitive fluorometers.
U.S. Pat. No. 3,992,631 describes a sensitive fluorometer useful in fluorescent assays. Highly suitable fluorescently labeled hormones have now been developed and have been found to work satisfactorily in a competitive protein binding analysis.